Methods and Materials for GALGT2 Gene Therapy

ABSTRACT

The present disclosure relates to recombinant adeno-associated virus (rAAV) delivery of a GALGT2 polynucleotide. The disclosure provides rAAV and methods of using the rAAV for GALGT2 gene therapy of neuromuscular disorders. Exemplary neuromuscular disorders include, but are not limited to, muscular dystrophies such as Duchenne muscular dystrophy, Congenital Muscular Dystrophy 1A and Limb Girdle Muscular Dystrophy 2D.

This application is a continuation of U.S. patent application Ser. No.15/759,474, filed Mar. 12, 2018, which is a national phase applicationof PCT Application No. PCT/US16/52051, filed Sep. 16, 2016, which claimspriority benefit of U.S. Provisional Application No. 62/301,260, filedFeb. 29, 2016, U.S. Provisional Application No. 62/221,068, filed onSep. 20, 2015 and U.S. Provisional Application No. 62/220,107, filedSep. 17, 2015, all of which are incorporated by reference herein intheir entirety.

STATEMENT OF GOVERNMENT INTEREST

This invention was made with government support under U54 NS055958 andR01 AR049722 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

INCORPORATION BY REFERENCE OF THE SEQUENCE LISTING

This application contains, as a separate part of disclosure, a SequenceListing in computer-readable form (filename: 49985A_Seqlisting.txt;14,552 bytes—ASCII text file; created Mar. 16, 2021) which isincorporated by reference herein in its entirety.

FIELD

The present disclosure relates to recombinant adeno-associated virus(rAAV) delivery of a GALGT2 polynucleotide. The disclosure provides rAAVand methods of using the rAAV for GALGT2 gene therapy of neuromusculardisorders. Exemplary neuromuscular disorders include, but are notlimited, to muscular dystrophies such as Duchenne muscular dystrophy,Congenital Muscular Dystrophy 1A and Limb Girdle Muscular Dystrophy 2D.

BACKGROUND

Muscular dystrophies (MDs) are a group of genetic diseases. The group ischaracterized by progressive weakness and degeneration of the skeletalmuscles that control movement. Some forms of MD develop in infancy orchildhood, while others may not appear until middle age or later. Thedisorders differ in terms of the distribution and extent of muscleweakness (some forms of MD also affect cardiac muscle), the age ofonset, the rate of progression, and the pattern of inheritance.

One type of MD is Duchenne muscular dystrophy (DMD). It is the mostcommon severe childhood form of muscular dystrophy affecting 1 in 5000newborn males. Inheritance follows an X-linked recessive pattern. DMD iscaused by mutations in the DMD gene leading to absence of dystrophinprotein (427 KDa) in skeletal and cardiac muscles, as well as GI tractand retina. Dystrophin not only protects the sarcolemma from eccentriccontractions, but also anchors a number of signaling proteins in closeproximity to sarcolemma. Clinical symptoms of DMD are usually firstnoted between ages 3 to 5 years, with altered gait and reduced motorskills typically leading to diagnostic evaluation. DMD is relentlesslyprogressive, with loss of ambulation by age twelve. Historicallypatients died from respiratory complications late in the second decade,but improved supportive care—and in particular judicious use ofnocturnal ventilatory support—has extended life expectancy by nearly adecade. Prolonging life unmasks the nearly universal decline in cardiacfunction, with complications of dilated cardiomyopathy. This posesfurther clinical challenges and a need for recognition and medicalintervention that did not previously exist. Non-progressive cognitivedysfunction may also be present in DMD. Despite virtually hundreds ofclinical trials in DMD, treatment with corticosteroids remains the onlytreatment that has consistently demonstrated efficacy. Current standardof care for DMD involves use of prednisone or deflazacort, which canprolong ambulation by several years at the expense of significant sideeffects, and has limited evidence for any impact on survival.

Another type of MD is Congenital Muscular Dystrophy 1A (MCD1A). MCD1Abelongs to a group of neuromuscular disorders with onset at birth orinfancy characterized by hypotonia, muscle weakness and muscle wasting.MCD1A represents 30-40% of congenital muscular dystrophies, with someregional variation. Prevalence is estimated at 1/30,000. The diseasepresents at birth or in the first few months of life with hypotonia andmuscle weakness in the limbs and trunk. Respiratory and feedingdisorders can also occur. Motor development is delayed and limited(sitting or standing is only possible with help). Infants present withearly rigidity of the vertebral column, scoliosis, and respiratoryinsufficiency. There is facial involvement with a typical elongatedmyopathic face and ocular ophthalmoplegia disorders can appear later.Epileptic attacks are possible, although they occur in less than a thirdof subjects. Intellectual development is normal. MCD1A is caused bymutations in the LAMA2 gene coding for the alpha-2 laminin chain.Transmission is autosomal recessive. Current treatment is symptomatic.It consists of a multidisciplinary approach, including physiotherapists,occupational therapists and speech-language therapists, with theobjective of optimizing each subject's abilities. Seizures or otherneurological complications require specific treatment. The prognosis ofMDC1A is very severe as a large proportion of affected children do notreach adolescence. Currently, the prognosis can only be improved byattentive multidisciplinary (particularly orthopedic and respiratory)management.

Yet another type of MD is Limb Girdle Muscular Dystrophy (LGMD). LGMDsare rare conditions and they present differently in different peoplewith respect to age of onset, areas of muscle weakness, heart andrespiratory involvement, rate of progression and severity. LGMDs canbegin in childhood, adolescence, young adulthood or even later. Bothgenders are affected equally. LGMDs cause weakness in the shoulder andpelvic girdle, with nearby muscles in the upper legs and arms sometimesalso weakening with time. Weakness of the legs often appears before thatof the arms. Facial muscles are usually unaffected. As the conditionprogresses, people can have problems with walking and may need to use awheelchair over time. The involvement of shoulder and arm muscles canlead to difficulty in raising arms over head and in lifting objects. Insome types of LGMD, the heart and breathing muscles may be involved.

There are at least nineteen forms of LGMD, and the forms are classifiedby their associated genetic defects.

Type Pattern of Inheritance Gene or Chromosome LGMD1A Autosomal dominantMyotilin gene LGMD1B Autosomal dominant Lamin A/C gene LGMD1C Autosomaldominant Caveolin gene LGMD1D Autosomal dominant Chromosome 7 LGMD1EAutosomal dominant Desmin gene LGMD1F Autosomal dominant Chromosome 7LGMD1G Autosomal dominant Chromosome 4 LGMD1H Autosomal dominantChromosome 3 LGMD2A Autosomal recessive Calpain-3 gene LGMD2B Autosomalrecessive Dysferlin gene LGMD2C Autosomal recessive Gamma-sarcoglycangene LGMD2D Autosomal recessive Alpha-sarcoglycan gene LGMD2E Autosomalrecessive Beta-sarcoglycan gene LGMD2F Autosomal recessiveDelta-sarcoglycan gene LGMD2G Autosomal recessive Telethonin gene LGMD2HAutosomal recessive TRIM32 LGMD2I Autosomal recessive FKRP gene LGMD2JAutosomal recessive Titin gene LGMD2K Autosomal recessive POMT1 geneLGMD2L Autosomal recessive Fukutin gene LGMD2M Autosomal recessiveFukutin gene LGMD2N Autosomal recessive POMT2 gene LGMD2O Autosomalrecessive POMGnT1 gene LGMD2Q Autosomal recessive Plectin gene

Specialized tests for LGMD are now available through a national schemefor diagnosis, the National Commissioning Group (NCG).

The GALGT2 gene (otherwise known as B4GALNT2) encodes aβ1-4-N-acetyl-D-galactosamine (βGalNAc) glycosyltransferase. GALGT2overexpression has been studied in three different models of musculardystrophy: DMD, LGMD2D and MDC1A [Xu et al., Am. J. Pathol, 175: 235-247(2009); Xu et al., Am. J. Path., 171: 181-199 (2007); Xu et al.,Neuromuscul. Disord., 17: 209-220 (2007); Martin et al., Am. J. Physiol.Cell. Physiol., 296: C476-488 (2009); and Nguyen et al., Proc. Natl.Acad. Sci. USA, 99: 5616-5621 (2002)]. GALGT2 overexpression in skeletalmuscles has been reported to induce the glycosylation of alphadystroglycan with β1-4-N-acetyl-D-galactosamine (GalNAc) carbohydrate tomake the CT carbohydrate antigen(Neu5Ac/Gcα2-3[GalNAcβ1-4]Galβα1-4GlcNAcβ−). The GALGT2glycosyltransferase and the CT carbohydrate it creates are normallyconfined to neuromuscular and myotendinous junctions in skeletal musclesof adult humans, non-human primates, rodents and all other mammals yetstudied [Martin et al., J. Neurocytol., 32: 915-929 (2003)].Overexpression of GALGT2 in skeletal muscle has been reported tostimulate the ectopic glycosylation of the extrasynaptic membrane,stimulating the ectopic overexpression of a scaffold of normallysynaptic proteins that are orthologues or homologues of proteins missingin various forms of muscular dystrophy, including dystrophin surrogates(e.g., utrophin, Plectin1) and laminin α2 surrogates (laminin α5 andagrin) [Xu et al. 2009, supra; Xu et al, Am. J. Path. 2007, supra; Xu etal., Neuromuscul. Disord. 2007, supra; Nguyen et al., supra; Chicoine etal., Mol. Ther, 22: 713-724. (2014). As a group, the induction of suchsurrogates by GALGT2 has been reported to strengthen sarcolemmalmembrane integrity and prevent muscle injury in dystrophin-deficientmuscles as well as in wild type muscles [Martin et al., supra]. GALGT2overexpression in skeletal muscle has been reported to prevent muscledamage and inhibit muscle disease. This is true in the mdx mouse modelfor DMD [Xu et al., Neuromuscul. Disord. 2007, supra; Martin et al.(2009), supra; Nguyen et al., supra], where improvement equal to that ofmicro-dystrophin gene transfer is noted even though only half the numberof fibers were transduced [Martin et al. (2009), supra]. Notably, GALGT2gene transfer has also been reported to be preventive in the dy^(W)model for congenital muscular dystrophy 1A [Xu et al, Am. J. Path. 2007,supra] and the Sgca^(−/−) mouse model for limb girdle muscular dystrophytype 2D [Xu et al. 2009, supra].

Adeno-associated virus (AAV) is a replication-deficient parvovirus, thesingle-stranded DNA genome of which is about 4.7 kb in length including145 nucleotide inverted terminal repeat (ITRs). There are multipleserotypes of AAV. The nucleotide sequences of the genomes of the AAVserotypes are known. For example, the complete genome of AAV-1 isprovided in GenBank Accession No. NC_002077; the complete genome ofAAV-2 is provided in GenBank Accession No. NC_001401 and Srivastava etal., J. Virol., 45: 555-564 {1983); the complete genome of AAV-3 isprovided in GenBank Accession No. NC_1829; the complete genome of AAV-4is provided in GenBank Accession No. NC_001829; the AAV-5 genome isprovided in GenBank Accession No. AF085716; the complete genome of AAV-6is provided in GenBank Accession No. NC_00 1862; at least portions ofAAV-7 and AAV-8 genomes are provided in GenBank Accession Nos. AX753246and AX753249, respectively; the AAV-9 genome is provided in Gao et al.,J. Virol., 78: 6381-6388 (2004); the AAV-10 genome is provided in Mol.Ther., 13(1): 67-76 (2006); and the AAV-11 genome is provided inVirology, 330(2): 375-383 (2004). Cis-acting sequences directing viralDNA replication (rep), encapsidation/packaging and host cell chromosomeintegration are contained within the AAV ITRs. Three AAV promoters(named p5, p19, and p40 for their relative map locations) drive theexpression of the two AAV internal open reading frames encoding rep andcap genes. The two rep promoters (p5 and p19), coupled with thedifferential splicing of the single AAV intron (at nucleotides 2107 and2227), result in the production of four rep proteins (rep 78, rep 68,rep 52, and rep 40) from the rep gene. Rep proteins possess multipleenzymatic properties that are ultimately responsible for replicating theviral genome. The cap gene is expressed from the p40 promoter and itencodes the three capsid proteins VP1, VP2, and VP3. Alternativesplicing and non-consensus translational start sites are responsible forthe production of the three related capsid proteins. A single consensuspolyadenylation site is located at map position 95 of the AAV genome.The life cycle and genetics of AAV are reviewed in Muzyczka, CurrentTopics in Microbiology and Immunology, 158: 97-129 (1992).

AAV possesses unique features that make it attractive as a vector fordelivering foreign DNA to cells, for example, in gene therapy. AAVinfection of cells in culture is noncytopathic, and natural infection ofhumans and other animals is silent and asymptomatic. Moreover, AAVinfects many mammalian cells allowing the possibility of targeting manydifferent tissues in vivo. Moreover, AAV transduces slowly dividing andnon-dividing cells, and can persist essentially for the lifetime ofthose cells as a transcriptionally active nuclear episome(extrachromosomal element). The AAV proviral genome is infectious ascloned DNA in plasmids which makes construction of recombinant genomesfeasible. Furthermore, because the signals directing AAV replication,genome encapsidation and integration are contained within the ITRs ofthe AAV genome, some or all of the internal approximately 4.3 kb of thegenome (encoding replication and structural capsid proteins, rep-cap)may be replaced with foreign DNA. The rep and cap proteins may beprovided in trans. Another significant feature of AAV is that it is anextremely stable and hearty virus. It easily withstands the conditionsused to inactivate adenovirus (56° to 65° C. for several hours), makingcold preservation of AAV less critical. AAV may even be lyophilized.Finally, AAV-infected cells are not resistant to superinfection.

An AAV termed rh.74 has been used to deliver DNAs encoding variousproteins. Xu et al., Neuromuscular Disorders, 17: 209-220 (2007) andMartin et al., Am. J. Physiol. Cell. Physiol., 296: 476-488 (2009)relate to rh.74 expression of cytotoxic T cell GalNAc transferase forDuchenne muscular dystrophy. Rodino-Klapac et al., Mol. Ther., 18(1):109-117 (2010) describes AAV rh.74 expression of a micro-dystrophin FLAGprotein tag fusion after delivery of the AAV rh.74 by vascular limbperfusion.

The muscular dystrophies are a group of diseases without identifiabletreatment that gravely impact individuals, families, and communities.The costs are incalculable. Individuals suffer emotional strain andreduced quality of life associated with loss of self-esteem. Extremephysical challenges resulting from loss of limb function createshardships in activities of daily living. Family dynamics suffer throughfinancial loss and challenges to interpersonal relationships. Siblingsof the affected feel estranged, and strife between spouses often leadsto divorce, especially if responsibility for the muscular dystrophy canbe laid at the feet of one of the parental partners. The burden of questto find a cure often becomes a life-long, highly focused effort thatdetracts and challenges every aspect of life. Beyond the family, thecommunity bears a financial burden through the need for added facilitiesto accommodate the handicaps of the muscular dystrophy population inspecial education, special transportation, and costs for recurrenthospitalizations to treat recurrent respiratory tract infections andcardiac complications. Financial responsibilities are shared by stateand federal governmental agencies extending the responsibilities to thetaxpaying community.

There thus remains a significant need in the art for treatments forneuromuscular disorders including, but not limited to, musculardystrophies such as DMD, MDC1A and LGMD2D.

SUMMARY

Provided herein are methods of treating a neuromuscular disorder in asubject in need thereof in which a recombinant adeno-associated virus(rAAV) such as rAAVrh74.MCK.GALGT2 is administered to the subject,where: the route of administration is an intramuscular route and thedose of the rAAV administered is about 3×10¹¹ vg/injection to about5×10¹² vg/injection, the route of administration is an intramuscularroute and the dose of the rAAV administered is about 3×10¹¹vg/injection, the route of administration is an intramuscular route andthe dose of the rAAV administered is about 1×10¹² vg/injection, theroute of administration is an intramuscular route and the dose of therAAV administered is about 5×10¹² vg/injection, the route ofadministration is inter-arterial limb perfusion and the dose of the rAAVadministered is about 6×10¹² vg/kg/limb to about 4.8×10¹³ vg/kg/limb,the route of administration is inter-arterial limb perfusion and thedose of the rAAV administered is about 6×10¹² vg/kg/limb, the route ofadministration is inter-arterial limb perfusion and the dose of the rAAVadministered is about 1.2×10¹³ vg/kg/limb, the route of administrationis inter-arterial limb perfusion and the dose of the rAAV administeredis about 2.4×10¹³ vg/kg/limb, the route of administration isinter-arterial limb perfusion and the dose of the rAAV administered isabout 4.8×10¹³ vg/kg/limb, the route of administration is systemicintravenous administration and the dose of the rAAV administered isabout 2×10¹⁴ vg/kg to about 6×10¹⁵ vg/kg, the route of administration issystemic intravenous administration and the dose of the rAAVadministered is about 4×10¹⁴ vg/kg to about 6×10¹⁵ vg/kg, the route ofadministration is systemic intravenous administration and the dose ofthe rAAV administered is about 4×10¹⁴ vg/kg, the route of administrationis systemic intravenous administration and the dose of the rAAVadministered is about 8×10¹⁴ vg/kg, the route of administration issystemic intravenous administration and the dose of the rAAVadministered is about 2×10¹⁵ vg/kg or the route of administration issystemic intravenous administration and the dose of the rAAVadministered is about 6×10¹⁵ vg/kg.

Examples of neuromuscular disorder for which treatment is contemplatedare Duchenne Muscular Dystrophy (DMD); Becker Muscular Dystrophy;Congenital Muscular Dystrophy (MDC) 1A, 1B, 1C and 1D; Limb GirdleMuscular Dystrophy (LGMD) 1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 2A, 2B, 2C,2D, 2E, 2F, 2G 2H, 2I, 2J, 2K, 2L, 2M, 2N, 20 and 2Q; Bethlem Myopathy;Ullrich Congenital Muscular Dystrophy; Muscle Eye Brain Disease;Fukuyama Congenital Muscular Dystrophy; Walker Warburg Syndrome;Myotonic Dystrophy; Myasthenic syndromes; Congenital Myasthenias;Inclusion Body Myopathy; Inclusion Body Myositis; Emery DreifussMuscular Dystrophy; Distal Muscular Dystrophy; Dermatomyositis;Centronuclear Myopathy; Faciosacpulohumeral Muscular Dystrophy; MyoshiMyopathy; Mitochondrial Myopathy; Nemaline Myopathy; Nonaka Myopathy;Myasthenia Gravis; and Polymyositis. Thus, among others, musculardystrophies are neuromuscular disorders contemplated.

The methods result in an improvement in the human subject, for example,in absolute muscle specific force; force decrement during eccentricmuscle contractions; serum CK level; serum cardiac troponin level; serumMMP9 level; grip strength; limb torque; limb mobility or flexibility;ambulation; 6 minute walk test; knee flexor or extensor strength;maximal voluntary isometric muscle contraction; North Star AmbulatoryAssessment; muscle mass, fat reduction, or edema by limb T2-weighted MRImeasures; muscle contractures; limb joint angle; heart function (heartrate, cardiac output, percent fractional shortening, stroke volume);respiration (including respiratory rate, blood oxygenation, need forsupplemental oxygen); muscle necrosis; muscle regeneration; musclewasting; muscle inflammation; muscle calcification; muscle centralnucleation; muscle size or myofiber size; lifespan; and/or dystrophin orlaminin alpha 2 surrogate protein expression (utrophin, plectin 1,laminin alpha 5, agrin).

Also provided herein are rAAV encoding a GALGT2 polypeptide such as therAAVrh74.MCK.GALGT2.

DETAILED DESCRIPTION

The present disclosure provides methods and products for treatingneuromuscular disorders. The methods involve delivery of GALGT2polynucleotides to muscle cells in a subject using AAV as a genedelivery vector. Subjects include, but are not limited to, mammals suchas dogs, cats and humans. In some embodiments, the subjects are humanpatients. In some embodiments, the subjects are human pediatricpatients.

In one aspect, methods are provided for the treatment of neuromusculardisorders comprising administering to a subject a recombinant AAV (rAAV)encoding GALGT2.

Neuromuscular disorders contemplated herein include, but are not limitedto, a muscular dystrophy (MD). Neuromuscular disorders contemplatedherein, also include neuromuscular disorders other than MDs. Thus, insome embodiments, neuromuscular disorders contemplated herein include,but are not limited to, Duchenne Muscular Dystrophy (DMD); BeckerMuscular Dystrophy; Congenital Muscular Dystrophy (MDC) 1A, 1B, 1C and1D; Limb Girdle Muscular Dystrophy (LGMD) 1A, 1B, 1C, 1D, 1E, 1F, 1G,1H, 2A, 2B, 2C, 2D, 2E, 2F, 2G 2H, 2I, 2J, 2K, 2L, 2M, 2N, 20 and 2Q;Bethlem Myopathy; Ullrich Congenital Muscular Dystrophy; Muscle EyeBrain Disease; Fukuyama Congenital Muscular Dystrophy; Walker WarburgSyndrome; Myotonic Dystrophy; Myasthenic syndromes; CongenitalMyasthenias; Inclusion Body Myopathy; Inclusion Body Myositis; EmeryDreifuss Muscular Dystrophy; Distal Muscular Dystrophy; Dermatomyositis;Centronuclear Myopathy; Faciosacpulohumeral Muscular Dystrophy; MyoshiMyopathy; Mitochondrial Myopathy; Nemaline Myopathy; Nonaka Myopathy;Myasthenia Gravis; and Polymyositis. In some embodiments, the MD is DMD.In some embodiments, the MD is MDC1A. In some embodiments, the MD isLGMD2D.

In some embodiments of any of the methods described herein, the subjectis at least 1 year of age (e.g., at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19, or 20) or older. In some embodimentsof any of the methods described herein, the subject is a male. In someembodiments of any of the methods described herein, the subject is afemale. In some embodiments of any of the methods described herein, thesubject is ambulant at the time of treatment. In some embodiments of anyof the methods described herein, the subject is non-ambulant at the timethat treatment begins. In some embodiments of any of the methodsdescribed herein, the subject is one having a confirmed mutation in theDMD gene using a clinically accepted technique that defines themutation. Mutations in the dystrophin gene that give rise to a DMDphenotype are well known in the art, as are methods for identifying themin a subject. See, e.g., the Leiden Duchenne muscular dystrophy mutationdatabase, Leiden University Medical Center, The Netherlands, andAartsma-Rus et al., Hum. Mut., 30:293-299 (2009).

In some embodiments of any of the methods described herein, the subject,by magnetic resonance imaging of the extensor digitorum brevis (EDB)muscle, exhibits a preservation of sufficient muscle mass to permittransfection or gene transfer. In some embodiments of any of the methodsdescribed herein, the subject is receiving a stable dose ofcorticosteroid therapy (e.g., deflazacort, prednisone, or a generic formthereof), e.g., for at least 4 (e.g., 5, 6, 7, 8, 9, 10, 11, or 12)weeks prior to beginning treatment. In certain embodiments, the subjectis on background steroid therapy (e.g., intermittent orchronic/continuous background steroid therapy). One of skill in the artwould appreciate that such subjects are those who are subject to ongoinguse of steroids (or corticosteroids) on top of which another treatment,such as the gene therapy described herein, is administered.

In some embodiments of any of the methods described herein, the subjectis one that does not have an active viral infection. In some embodimentsof any of the methods described herein, the subject the subject does nothave a DMD mutation in the absence of weakness or loss of function. Insome embodiments of any of the methods described herein, the subjectdoes not exhibit symptoms of cardiomyopathy, such as dyspnea onexertion, pedal edema, shortness of breath upon lying flat, or rales atthe base of the lung. In some embodiments of any of the methodsdescribed herein, the subject does not, by echocardiogram, have anejection fraction below about 40%. In some embodiments of any of themethods described herein, the subject is not one for whom serologicalevidence exists at the time of treatment of infection with HIV (sincethe subject may be in an immunocompromised state) or Hepatitis A, B, orC (since the subject may have transaminase elevation).

In some embodiments of any of the methods described herein, the subjecthas not been diagnosed, does not have, or is not being treated for anautoimmune disease. In some embodiments of any of the methods describedherein, the subject does not have persistent leukopenia or leukocytosis(white blood cell count ≤3.5 K/μL or ≥20.0 K/μL) or an absoluteneutrophil count <1.5 K/μL.

In some embodiments of any of the methods described herein, the subjectdoes not have a concomitant illness (e.g., viral infection or autoimmunedisease) or requirement for chronic drug treatment that in the opinionof a medical practitioner creates an unnecessary risk for gene transfer.

In some embodiments of any of the methods described herein, the subjectdoes not have an rAAVrh74 binding antibody titer of ≤1:400 as determinedby an ELISA immunoassay. In some embodiments of any of the methodsdescribed herein, the subject does not have an rAAVrh74 binding antibodytiter of ≤1:50 as determined by an ELISA immunoassay.

In some embodiments of any of the methods described herein, the subjectdoes not have detectable circulating anti-Sda antibodies, e.g., asdetermined by ELISA immunoassay of a biological sample of the subject[Blood groups: P, I, Sda, and Pr. AABB (1991)]. The Sda glycan is thehuman blood group structure made by GALGT2. The Sda blood group antigenis identical to the CT glycan structure made by GALGT2 in mice[GalNAcb1,4[Neu5Aca2,3]Galb1,4GlcNAc−]. While the disclosure is notbound by any particular theory or mechanism of action, the presence oramount of the CT glycan structure is expected to be enhanced on cellsexpressing GALGT2 and the inventors believe that subjects with such Sdaantibodies, estimated to be 0.2% of the subject population, may be atrisk of antibody-mediated tissue rejection after rAAVrh74.MCK.GALGT2treatment.

Suitable biological samples for use in the methods described hereininclude, e.g., any biological fluid. A biological sample can be, forexample, a specimen obtained from a subject (e.g., a mammal such as ahuman) or can be derived from such a subject. A biological sample can befrom a muscle biopsy. A biological sample can also be a biological fluidsuch as urine, whole blood or a fraction thereof (e.g., plasma orserum), saliva, semen, sputum, cerebrospinal fluid, tears, or mucus. Abiological sample can be further fractionated, if desired, to a fractioncontaining particular analytes (e.g., proteins) of interest. Forexample, a whole blood sample can be fractionated into serum or intofractions containing particular types of proteins. If desired, abiological sample can be a combination of different biological samplesfrom a subject such as a combination of two different fluids.

Biological samples suitable for the invention may be fresh or frozensamples collected from a subject, or archival samples with knowndiagnosis, treatment and/or outcome history. The biological samples canbe obtained from a subject, e.g., a subject having, suspected of having,or at risk of developing, a cancer or an infection (e.g., a viralinfection). Any suitable methods for obtaining the biological samplescan be employed, although exemplary methods include, for example, openmuscle biopsy, phlebotomy, swab (e.g., buccal swab), lavage, or fineneedle aspirate biopsy procedure. Biological samples can also beobtained from bone marrow or spleen.

In some embodiments, a protein extract may be prepared from a biologicalsample. In some embodiments, a protein extract contains the totalprotein content. Methods of protein extraction are well known in theart. See, for example, Roe “Protein Purification Techniques: A PracticalApproach”, 2nd Edition, Oxford University Press (2001). Numerousdifferent and versatile kits can be used to extract proteins from bodilyfluids and tissues, and are commercially-available from, for example,BioRad Laboratories (Hercules, Calif.), BD Biosciences Clontech(Mountain View, Calif.), Chemicon International, Inc. (Temecula,Calif.), Calbiochem (San Diego, Calif.), Pierce Biotechnology (Rockford,Ill.), and Invitrogen Corp. (Carlsbad, Calif.).

Methods for obtaining and/or storing samples that preserve the activityor integrity of cells in the biological sample are well known to thoseskilled in the art. For example, a biological sample can be furthercontacted with one or more additional agents such as appropriate buffersand/or inhibitors, including protease inhibitors, the agents meant topreserve or minimize changes (e.g., changes in osmolarity or pH) inprotein structure. Such inhibitors include, for example, chelators suchas ethylenediamine tetraacetic acid (EDTA), ethylene glycol tetraaceticacid (EGTA), protease inhibitors such as phenylmethylsulfonyl fluoride(PMSF), aprotinin, and leupeptin. Appropriate buffers and conditions forstoring or otherwise manipulating whole cells are described in, forexample, Pollard and Walker, “Basic Cell Culture Protocols,” Volume 75of Methods in Molecular Biology, Humana Press (1997); Masters, “Animalcell culture: a practical approach,” Volume 232 of Practical ApproachSeries, Oxford University Press (2000); and Jones “Human cell cultureprotocols,” Volume 2 of Methods in Molecular Medicine, Humana Press(1996).

The methods of treating neuromuscular disorders disclosed herein resultin transduction of muscle cells (e.g., skeletal muscle, smooth muscle orcardiac muscle cells) with GALGT2 polynucleotide. An effective dose, oreffective multiple doses, of a composition comprising a rAAV of thedisclosure to a subject is a dose that prevents, slows progression of,or ameliorates (eliminates or reduces) muscle pathology associated withthe neuromuscular disorder being treated. An effect on muscle pathologycan be demonstrated by an improvement in one or more measures standardin the art such as: absolute muscle specific force; force decrementduring eccentric muscle contractions; serum CK level; serum cardiactroponin level; serum MMP9 level; grip strength; limb torque; limbmobility or flexibility; ambulation; 6 minute walk test; knee flexor orextensor strength; maximal voluntary isometric muscle contraction; NorthStar Ambulatory Assessment; muscle mass, fat reduction, or edema by limbT2-weighted MRI measures; muscle contractures; limb joint angle; heartfunction (heart rate, cardiac output, percent fractional shortening,stroke volume); respiration (including respiratory rate, bloodoxygenation, need for supplemental oxygen); muscle necrosis; muscleregeneration; muscle wasting; muscle inflammation; muscle calcification;muscle central nucleation; muscle size or myofiber size; lifespan; anddystrophin or laminin alpha 2 surrogate protein expression (utrophin,plectin 1, laminin alpha 5, agrin). See, for example, Forbes et al.,Radiology, 269(1): 198-207 (2013); Govoni et al., Cell Mol. Life Sci.,70(23): 4585-4602 (2013); and Chandrasekharan and Martin, MethodsEnzymol., 479: 291-322 (2010). If a dose is administered prior todevelopment of a neuromuscular disorder, the administration isprophylactic. If a dose is administered after the development of aneuromuscular disorder, the administration is therapeutic. The treatmentof the subject by methods described herein is therefore contemplated toprevent, slow or prevent progression of, diminish the extent of, resultin remission (partial or total) of, and/or prolong survival of aneuromuscular disorder.

In some embodiments, any of the methods described herein can furthercomprise detecting or measuring the level of expression of GALGT2 incells transduced with the GALGT2 transgene. Methods for measuring mRNAor protein expression are well known in the art (e.g., immunoassays,such as Western blotting).

In some embodiments, any of the methods described herein can furthercomprise detecting or measuring the amount of CT antigen expressed oncells transduced with the GALGT2 polynucleotide [Chicoine et al.,supra].

In some embodiments, any of the methods described herein can furthercomprise detecting or measuring the amount of utrophin expressed oncells transduced with the GALGT2 polynucleotide [Chicoine et al.,supra].

In some embodiments, any of the methods described herein can furthercomprise detecting or measuring the number of fibers containing centralnuclei, which fibers were transduced with the GALGT2 polynucleotide.

Routes of administration for the rAAV contemplated in the foregoingmethods therefore include, but are not limited to, intraperitoneal (IP),intramuscular (IM) and intravascular [including, for example,inter-arterial limb perfusion (ILP) and intravenous (IV)] routes.

The dose of rAAV to be administered in methods disclosed herein willvary depending, for example, on the particular rAAV, the mode ofadministration, the treatment goal, the individual, and the cell type(s)being targeted, and may be determined by methods standard in the art.More than one dose may be administered, for example, one, two, three ormore doses. Titers of rAAV in a dose may range from about 1×10⁶, about1×10⁷, about 1×10⁸, about 1×10⁹, about 1×10¹⁰, about 1×10¹¹, about1×10¹², about 1×10¹³, about 1×10¹⁴, or to about 1×10¹⁵ or more DNaseresistant particles (DRP) per ml. Dosages may also be expressed in unitsof viral genomes (vg) (i.e., 1×10⁷ vg, 1×10⁸ vg, 1×10⁹ vg, 1×10¹⁰ vg,1×10¹¹ vg, 1×10¹² vg, 1×10¹³ vg, 1×10¹⁴ vg, 1×10¹⁵ respectively).Methods for tittering AAV are described in Clark et al., Hum. GeneTher., 10: 1031-1039 (1999).

In some embodiments of the foregoing methods in which the route ofadministration is an IM route, the dose of the rAAV administered is fromabout 3×10¹¹ to at least about 5×10¹² vg/injection. (All ranges hereinare intended to represent each individual value in the ranges, as wellas the individual upper and lower values of each range.) In someembodiments of the foregoing methods in which the route ofadministration is an IM route, the dose of the rAAV administered is3×10¹¹ vg/injection. In some embodiments of the foregoing methods inwhich the route of administration is an IM route, the dose of the rAAVadministered is 1×10¹² vg/injection. In some embodiments of theforegoing methods in which the route of administration is an IM route,the dose of the rAAV administered is 5×10¹² vg/injection.

In some embodiments of the foregoing methods in which the route ofadministration is an ILP route, the dose of the rAAV administered isfrom about 6×10¹² to at least about 4.8×10¹³ vg/kg. (All ranges hereinare intended to represent each individual value in the ranges, as wellas the individual upper and lower values of each range.) In someembodiments of the foregoing methods in which the route ofadministration is ILP, the dose of the rAAV administered is 6×10¹²vg/kg/limb. In some embodiments of the foregoing methods in which theroute of administration is ILP, the dose of the rAAV administered is1.2×10¹³ vg/kg/limb. In some embodiments of the foregoing methods inwhich the route of administration is ILP, the dose of the rAAVadministered is 2.4×10¹³ vg/kg/limb. In some embodiments of theforegoing methods in which the route of administration is ILP, the doseof the rAAV administered is 4.8×10¹³ vg/kg/limb.

In some embodiments of the foregoing methods in which the route ofadministration is a systemic IV route, the dose of the rAAV administeredis from about 2×10¹⁴ to at least about 6×10¹⁵ vg/kg. In some embodimentsof the foregoing methods in which the route of administration is asystemic IV route, the dose of the rAAV administered is from about4×10¹⁴ to at least about 6×10¹⁵ vg/kg. (All ranges herein are intendedto represent each individual value in the ranges, as well as theindividual upper and lower values of each range.) In some embodiments ofthe foregoing methods in which the route of administration is systemicIV administration, the dose of the rAAV administered is 4×10¹⁴ vg/kg. Insome embodiments of the foregoing methods in which the route ofadministration is systemic IV administration, the dose of the rAAVadministered is 8×10¹⁴ vg/kg. In some embodiments of the foregoingmethods in which the route of administration is systemic IVadministration, the dose of the rAAV administered is 2×10¹⁵ vg/kg. Insome embodiments of the foregoing methods in which the route ofadministration is systemic IV administration, the dose of the rAAVadministered is 6×10¹⁵ vg/kg.

Human patients are subjects contemplated herein for treatment. Humanpatients are subjects contemplated herein for treatment by IM delivery.Such patients include those patients that, e.g.: (i) are 9 years of ageor older, (ii) are male, (iii) are ambulant or non-ambulant; (iv) have aconfirmed mutation in the DMD gene using a clinically accepted techniquethat defines the mutation; (v) by magnetic resonance imaging of theextensor digitorum brevis (EDB) muscle show a preservation of sufficientmuscle mass to permit transfection or gene transfer; (vi) are of anyethnic group; (vii) have the ability to cooperate with all studyprocedures; (viii), if appropriate, and sexually mature and/or active,are willing to practice a reliable method of contraception; and/or (ix)are receiving a stable dose of corticosteroid therapy (e.g.,deflazacort, prednisone, or a generic form thereof) for at least 12weeks prior to gene transfer. Suitable patients may not include, e.g.,those: (i) with active viral infections based on clinical observation;(ii) who have a DMD mutation without weakness or loss of function; (iii)with symptoms of cardiomyopathy, such as dyspnea on exertion, pedaledema, shortness of breath upon lying flat, or rales at the base of thelung; (iv) who, by echocardiogram, have an ejection fraction below about40%; (v) with serological evidence of infection with HIV or Hepatitis A,B, or C; (vi) who have or have been diagnosed as having (or are beingtreated for) an autoimmune disease; (vii) who have persistent leukopeniaor leukocytosis (white blood cell count ≤3.5 K/μL or ≥20.0 K/μL) or anabsolute neutrophil count <1.5 K/μL; (viii) who have a concomitantillness or requirement for chronic drug treatment that in the opinion ofa medical practitioner creates an unnecessary risk for gene transfer;(ix) who have an rAAVrh74 binding antibody titer of ≥1:400 as determinedby an ELISA immunoassay; or (x) have the presence of circulatinganti-Sda antibodies. In an exemplary clinical protocol, DMD patientsreceive bilateral injections with one extensor digitorum brevis (EDB)muscle of each patient receiving the vector rAAVrh74.MCK.GALGT2 and theother EDB muscle of each patient receiving saline alone. Subjectsreceive a dose of vector of 1×10¹² vg (total dose).

Cell transduction efficiencies of the methods described above and belowmay be at least about 60, 65, 70, 75, 80, 85, 90, or 95 percent. In someembodiments involving IV limb perfusion delivery, transductionefficiency is increased by increasing the volume of the composition inwhich the rAAV is delivered, pre-flushing before delivery of the rAAVand/or increasing dwell time of the rAAV.

In another aspect, rAAV genomes are provided herein. The genomes of therAAV administered comprise a GALGT2 polynucleotide under the control oftranscription control sequences. The rAAV genomes lack AAV rep and capDNA. AAV DNA in the rAAV genomes may be from any AAV serotype for whicha recombinant virus can be derived including, but not limited to, AAVserotypes AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9,AAV-10, AAV-11 and AAVrh.74. The nucleotide sequences of the genomes ofthese AAV serotypes are known in the art as noted in the BackgroundSection above. In some embodiments, the AAV DNA in the rAAV genomes isfrom AAV rh.74. The polynucleotide sequence of the AAV rh.74 genome isset out in SEQ ID NO: 1, wherein nucleotides 210-2147 are the Rep 78gene open reading frame, 882-208 are the Rep52 open reading frame,2079-2081 are the Rep78 stop, 2145-2147 are the Rep78 stop, 1797-1800are a splice donor site, 2094-2097 are a splice acceptor site, 2121-2124are a splice acceptor site, 174-181 are the p5 promoter+1 predicted,145-151 are the p5 TATA box, 758-761 are the p19 promoter+1 predicted,732-738 are the p19 TATA box, 1711-1716 are the p40 TATA box, 2098-4314are the VP1 Cap gene open reading frame, 2509-2511 are the VP2 start,2707-2709 are the VP3 start and 4328-4333 are a polyA signal.

In some embodiments, the transcription control sequences of the rAAVgenomes are muscle-specific control elements, including, but not limitedto, those derived from the actin and myosin gene families, such as fromthe myoD gene family [See Weintraub et al., Science, 251: 761-766(1991)], the myocyte-specific enhancer binding factor MEF-2 [Cserjesiand Olson, Mol. Cell. Biol., 11: 4854-4862 (1991)], control elementsderived from the human skeletal actin gene [Muscat et al., Mol. Cell.Biol., 7: 4089-4099 (1987)], the cardiac actin gene, muscle creatinekinase (MCK) promoter [Johnson et al., Mol. Cell. Biol., 9:3393-3399(1989)] and the MCK enhancer, MHCK7 promoter (a modified version of MCKpromoter that incorporates an enhancer from myosin heavy chain [Salva etal., Mol. Ther., 15: 320-329 (2007)]), desmin promoter, control elementsderived from the skeletal fast-twitch troponin C gene, the slow-twitchcardiac troponin C gene and the slow-twitch troponin I gene:hypozia-inducible nuclear factors [Semenza et al., Proc. Natl. Acad.Sci. USA, 88: 5680-5684 (1991)], steroid-inducible elements andpromoters including the glucocorticoid response element (GRE) [See Maderand White, Proc. Natl. Acad. Sci. USA, 90: 5603-5607 (1993)], and othercontrol elements. In some embodiments, the transcription controlelements include the MCK promoter. In some embodiments, thetranscription control elements include the MHCK7 promoter.

In some embodiments, the GALGT2 polynucleotide in a rAAV genome is theGALGT2 cDNA set out in Genbank Accession #AJ517771 (set out asnucleotides 1002-2522 of SEQ ID NO: 2). In some embodiments, the GALGT2polynucleotide in a rAAV genome is the GALGT2 cDNA set out in GenbankAccession #AJ517771, or is a variant polynucleotide having 80%, 81%,82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%, 97%, 98% or 99% sequence identity to the GALGT2 cDNA. In someembodiments, the variant GALGT2 polynucleotide encodes the same GALGT2polypeptide as the polypeptide encoded by GALGT2 cDNA set out in GenbankAccession #AJ517771. The amino acid sequence of the GALGT2 polypeptideencoded by the GALGT2 cDNA set out in Genbank Accession #AJ517771 is setout in SEQ ID NO: 3. In some embodiments, the variant GALGT2polynucleotide encodes a variant GALGT2 polypeptide with at least oneamino acid sequence alteration as compared to the amino acid sequence ofthe polypeptide (SEQ ID NO: 3) encoded by GALGT2 cDNA set out in GenbankAccession #AJ517771. An amino acid sequence alteration can be, forexample, a substitution, a deletion, or an insertion of one or moreamino acids, preferably conservative substitutions. A variant GALGT2polypeptide can have any combination of amino acid substitutions,deletions or insertions where the glycosyltransferase activity of thepolypeptide is retained. In one aspect, a variant GALGT2 polypeptide canhave a number of amino acid alterations such that its amino acidsequence shares at least 60, 70, 80, 85, 90, 95, 97, 98, 99 or 99.5%identity with the amino acid sequence (SEQ ID NO: 3) encoded by GALGT2cDNA set out in Genbank Accession #AJ517771.

In some embodiments, the rAAV genome is the MCK.GALGT2 genome, thesequence of the GALGT2 gene cassette of which is set out in SEQ ID NO: 2and is annotated as follows.

STARTING ENDING NUCLEO- NUCLEO- TIDE TIDE NAME DESCRIPTION 53 230 5′ITRWild Type AAV2 inverted terminal repeat 236 442 MCK enhancer Mousemuscle creatine kinase enhancer 443 793 MCK core Mouse muscle creatinepromoter kinase core promoter 794 846 Mu MCK Native transcriptionalstart Exon 1 site of exon 1 of mouse MCK gene (untranslated) 847 943SV40 intron SV40 late 16S/19S splice donor and acceptor sites 944 10005′ untranslated 5′ untranslated region from region plasmid pCMVb 10022522 Human GALGT2 Human GALGT2 cDNA cDNA 2531 2579 Syn pA Artificialpolyadenylation signal 2581 2762 3′ ITR Wild Type AAV2 inverted terminalrepeat

In yet another aspect, an isolated nucleic acid comprising thenucleotide sequence depicted in SEQ ID NO: 2 is provided. In someembodiments, the isolated nucleic acid consists of the nucleotidesequence depicted in SEQ ID NO: 2.

Also provided is an isolated nucleic acid comprising, in order from 5′to 3′: (i) a first AAV2 inverted terminal repeat sequence (ITR); (ii) amuscle creatine kinase promoter sequence; (iii) a nucleotide sequenceencoding a human GALGT2 polypeptide; and (iv) a second AAV2 ITRsequence, wherein the human GALGT2 polypeptide has an amino acidsequence that is at least 90% identical to SEQ ID NO:3, is 100%identical to SEQ ID NO:3, or is encoded by nucleotides 1002-2522 of SEQID NO: 2.

Recombinant AAV comprising the foregoing nucleic acids are contemplatedas well as rAAV comprising a nucleotide sequence that is at least 90%identical to the nucleotide sequence depicted in SEQ ID NO:2.

DNA plasmids comprising rAAV genomes of the disclosure are provided. TheDNA plasmids comprise rAAV genomes contemplated herein. The DNA plasmidsare transferred to cells permissible for infection with a helper virusof AAV (e.g., adenovirus, E1-deleted adenovirus or herpesvirus) forassembly of the rAAV genome into infectious viral particles. Techniquesto produce rAAV particles, in which an AAV genome to be packaged, repand cap genes, and helper virus functions are provided to a cell arestandard in the art. Production of rAAV requires that the followingcomponents are present within a single cell (denoted herein as apackaging cell): a rAAV genome, AAV rep and cap genes separate from(i.e., not in) the rAAV genome, and helper virus functions. The AAV repand cap genes may be from any AAV serotype for which recombinant viruscan be derived and may be from a different AAV serotype than the rAAVgenome ITRs, including, but not limited to, AAV serotypes AAV-1, AAV-2,AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8, AAV-9, AAV-10, AAV-11 and AAVrh74. Production of pseudotyped rAAV is disclosed in, for example, WO01/83692. Other types of rAAV variants, for example rAAV with capsidmutations, are also contemplated. See, for example, Marsic et al.,Molecular Therapy, 22(11): 1900-1909 (2014).

A method of generating a packaging cell is to create a cell line thatstably expresses all the necessary components for AAV particleproduction. For example, a plasmid (or multiple plasmids) comprising arAAV genome lacking AAV rep and cap genes, AAV rep and cap genesseparate from the rAAV genome, and a selectable marker, such as aneomycin resistance gene, are integrated into the genome of a cell. AAVgenomes have been introduced into bacterial plasmids by procedures suchas GC tailing (Samulski et al., 1982, Proc. Natl. Acad. S6. USA,79:2077-2081), addition of synthetic linkers containing restrictionendonuclease cleavage sites (Laughlin et al., 1983, Gene, 23:65-73) orby direct, blunt-end ligation (Senapathy & Carter, 1984, J. Biol. Chem.,259:4661-4666). The packaging cell line is then infected with a helpervirus such as adenovirus. The advantages of this method are that thecells are selectable and are suitable for large-scale production ofrAAV. Other examples of suitable methods employ adenovirus orbaculovirus rather than plasmids to introduce rAAV genomes and/or repand cap genes into packaging cells. Methods for producing rAAV withself-complementary genomes are also known in the art.

General principles of rAAV production are reviewed in, for example,Carter, 1992, Current Opinions in Biotechnology, 1533-539; and Muzyczka,1992, Curr. Topics in Microbial. and Immunol., 158:97-129). Variousapproaches are described in Ratschin et al., Mol. Cell. Biol. 4:2072(1984); Hermonat et al., Proc. Natl. Acad. Sci. USA, 81:6466 (1984);Tratschin et al., Mol. Cell. Biol. 5:3251 (1985); McLaughlin et al., J.Virol., 62:1963 (1988); and Lebkowski et al., 1988 Mol. Cell. Biol.,7:349 (1988). Samulski et al. (1989, J. Virol., 63:3822-3828); U.S. Pat.No. 5,173,414; WO 95/13365 and corresponding U.S. Pat. No. 5,658,776; WO95/13392; WO 96/17947; PCT/US98/18600; WO 97/09441 (PCT/US96/14423); WO97/08298 (PCT/US96/13872); WO 97/21825 (PCT/US96/20777); WO 97/06243(PCT/FR96/01064); WO 99/11764; Perrin et al. (1995) Vaccine13:1244-1250; Paul et al. (1993) Human Gene Therapy 4:609-615; Clark etal. (1996) Gene Therapy 3:1124-1132; U.S. Pat. Nos. 5,786,211;5,871,982; and 6,258,595. The foregoing documents are herebyincorporated by reference in their entirety herein, with particularemphasis on those sections of the documents relating to rAAV production.

In a further aspect, the disclosure thus provides packaging cells thatproduce infectious rAAV. In one embodiment packaging cells may be stablytransformed cancer cells such as HeLa cells, 293 cells and PerC.6 cells(a cognate 293 line). In another embodiment, packaging cells are cellsthat are not transformed cancer cells, such as low passage 293 cells(human fetal kidney cells transformed with E1 of adenovirus), MRC-5cells (human fetal fibroblasts), WI-38 cells (human fetal fibroblasts),Vero cells (monkey kidney cells) and FRhL-2 cells (rhesus fetal lungcells).

The rAAV may be purified by methods standard in the art such as bycolumn chromatography or cesium chloride gradients. Methods forpurifying rAAV vectors from helper virus are known in the art andinclude methods disclosed in, for example, Clark et al., Hum. GeneTher., 10(6): 1031-1039 (1999); Schenpp and Clark, Methods Mol. Med., 69427-443 (2002); U.S. Pat. No. 6,566,118 and WO 98/09657.

Thus, in another aspect, the disclosure contemplates a rAAV comprising aGALGT2 polynucleotide. In some embodiments, the rAAV comprises AAV rh74capsid and a GALGT2 polynucleotide. In some embodiments, the genome ofthe rAAV lacks AAV rep and cap DNA. In some embodiments of the methods,the rAAV is rAAVrh74.MCK.GALGT2. In some embodiments, the rAAV is aself-complementary genome.

In another aspect, the disclosure contemplates compositions comprising arAAV described herein. Compositions of the disclosure comprise rAAV in apharmaceutically acceptable carrier. The compositions may also compriseother ingredients such as diluents. Acceptable carriers and diluents arenontoxic to recipients and are preferably inert at the dosages andconcentrations employed, and include buffers such as phosphate, citrate,or other organic acids; antioxidants such as ascorbic acid; lowmolecular weight polypeptides; proteins, such as serum albumin, gelatin,or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone;amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides, and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as Tween, pluronics orpolyethylene glycol (PEG). In some embodiments, the rAAV is formulatedin Tris, MgCl₂, NaCl and pluronic F68. In some embodiments, the rAAV isformulated in 20 mM Tris (pH 8.0), 1 mM MgCl₂ and 200 mM NaCl containing0.001% pluronic F68.

Combination treatments are also contemplated herein. Combinations asused herein include simultaneous treatment or sequential treatments.Combinations of methods of the disclosure with standard medicaltreatments (e.g., corticosteroids and/or immunosuppressive drugs) arespecifically contemplated, as are combinations with novel treatments. Invarious embodiments, subjects are treated with corticosteroids before,during or after (or with any permutation of combinations of two or moreof the three possibilities), the subject is treated according to amethod contemplated herein.

Sterile injectable solutions are prepared by incorporating rAAV in therequired amount in the appropriate solvent with various otheringredients enumerated above, as required, followed by filtersterilization. Generally, dispersions are prepared by incorporating thesterilized active ingredient into a sterile vehicle which contains thebasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders for the preparation ofsterile injectable solutions, the preferred methods of preparation arevacuum drying and the freeze drying technique that yield a powder of theactive ingredient plus any additional desired ingredient from thepreviously sterile-filtered solution thereof.

DESCRIPTION OF DRAWING

FIG. 1 provides a schematic of The rAAVrh74.MCK.GALGT2 gene cassettedescribed in Example 1.

EXAMPLES

Thus, aspects and embodiments of the invention are illustrated by thefollowing examples.

Example 1

A non-replicating rAAV termed rAAVrh74.MCK.GALGT2 was generated. TherAAV vector contains the complete human GALGT2 cDNA (Genbank Accession#AJ517771) under the control of a muscle creatine kinase promoter (MCK;a muscle specific promoter). A MCK promoter/enhancer sequence was usedto drive muscle-specific gene (expression and is composed of the mouseMCK core enhancer (206 bp) fused to the 351 bp MCK core promoter(proximal). After the core promoter, the 53 bp endogenous mouse MCKExon1 (untranslated) is present for efficient transcription initiation,followed by the SV40 late 16S/19S splice signals (97 bp) and a small5′UTR (61 bp). The intron and 5′ UTR are derived from plasmid pCMVB(Clontech). The GALGT2 cassette has a consensus Kozak sequenceimmediately in front of the ATG start and a small 53 bp synthetic polyAsignal for mRNA termination. The human GALGT2 cassette was previouslydescribed by Martin et al (2009), supra. The only viral sequencesincluded are the inverted terminal repeats (ITR) of AAV2, which arerequired for both viral DNA replication and packaging. ThepAAVrh74.MCK.GALGT2 plasmid contains the human GALGT2 cDNA expressioncassette flanked by AAV2 inverted terminal repeat (ITR) sequences. Thegene cassette includes an MCK promoter, a chimeric intron with a Kozaksequence for optimizing gene expression, human GALGT2 coding sequencesand a polyA signal (see FIG. 1). The sequence of the gene cassette withflanking AAV ITRs is set out in SEQ ID NO: 2.

The AAV vectors including the GALGT2 polynucleotides were produced by amodified cross-packaging approach in an adenovirus-free, triple plasmidDNA transfection (CaPO4 precipitation) method in HEK293 cells[Rabinowitz et al., J. Virol., 76:791-801 (2002)]. Vector was producedby co-transfecting plasmid containing GALGT2 polynucleotide with an AAVhelper plasmid rep2-cap rh.74 and an adenovirus helper plasmid insimilar fashion as that previously described [Wang et al., Gene. Ther.,10:1528-1534 (2003)]. Plasmid rep2-cap rh.74 encodes the wild-type AAV2rep gene and rh.74 cap gene, and the adenovirus helper plasmid(pAdhelper) expresses the adenovirus type 5 E2A, E4ORF6, and VA I/II RNAgenes which are required for high-titer rAAV production.

Vectors were purified from clarified 293 cell lysates by sequentialiodixanol gradient purification and anion-exchange column chromatographyusing a linear NaCl salt gradient as previously described [Clark et al.,Hum. Gene Ther, 10:1031-1039 (1999)]. Vector genome (vg) titers weremeasured using QPCR based detection with a MCK specific primer/probe setand utilized the Prism 7500 Taqman detector system (PE AppliedBiosystems) as previously described (Clark et al., supra). Vector stocktiters ranged between 1-40×10¹² vg/mL.

The vector is formulated in 20 mM Tris (pH 8.0), 1 mM MgCl₂ and 200 mMNaCl containing 0.001% pluronic F68. The vector is supplied as a frozenliquid that is thawed before clinical administration.

Example 2 IM Delivery to Subjects with DMD

Human patients are subjects contemplated herein for treatment by IMdelivery. In an exemplary clinical protocol, DMD patients receivebilateral injections with one extensor digitorum brevis (EDB) muscle ofeach patient receiving the vector rAAVrh74.MCK.GALGT2 and the other EDBmuscle of each patient receiving saline alone. Subjects in a firstcohort receive a low dose of vector of 3×10¹¹ vg (total dose). Subjectsin a second cohort receive a higher dose of vector of 1×10¹² vg (totaldose).

Immediately prior to transportation to the clinical setting, appropriatedilutions of the vector are prepared. The dilution for the injection is1:1 with normal saline. The vector is kept on ice (not frozen) untiladministration and is administered to the subject within 8 hours ofpreparation. Handling of rAAVrh74.MCK.GALGT2 follows compliancestandards for Biosafety Level 1 vectors. See,www4.od.nih.gov/oba/RAC/guidelines 02/APPENDIX_G.htm#_Toc7246561.

Subjects have muscle weakness by clinical exam. The genetic diagnosis ofDMD is established on the basis of a DMD gene mutation consistent withDMD, in the setting of an appropriate clinical history. In this study,all subjects are non-ambulant, having lost ambulation in an age rangediagnostic of DMD (i.e., less than 12 years old without steroid therapy,or less than 15 years old in the setting of longstanding steroidtreatment). Subjects receive a stable dose of corticosteroid therapy(either prednisone or deflazacort, or their generic forms) for twelveweeks prior to treatment.

The vector or control is delivered via direct intramuscular injectioninto the extensor digitorum brevis (EDB) muscle of one foot of asubject, while the other foot receives saline alone. Conscious sedationis used on participants under 12 years of age. Patients over 12 years ofage may receive conscious sedation or a sedative (like lorazepam) atleast one hour prior to gene transfer. In addition, the skin over thegene transfer site is pre-treated with a lidocaine/prilocaine eutecticmixture incorporated in a cream base (EMLA cream) or a cellulose disk(EMLA patch). Comparable cream-based anesthesia such as xylocaine creammay be used. Procedures are performed under sterile conditions. Theinjection site is cleansed with three successive applications ofnon-iodine containing surgical prep swabs and draped with disposablesterile drapes. A standard clinical Doppler ultrasound is used with asterile sheath around the transducer to maintain asepsis of theinjection field. For vector injections of rAAVrh74.MCK.GALGT2 or placeboto the EDB, disposable MyoJect Needles that enable simultaneous EMGrecording and fluid injection are used to increase the precision ofmuscle injection. The anatomical midline point of the muscle isidentified on the skin and 2 to 6 separate vector injections aredistributed into the muscle. The injections are 0.5 cm in depth from themuscle surface. The total dose of vector is 3×10¹¹ vg in 1.5 ml in thelow dose group and 1×10¹² vg in 1.5 ml in the high-dose group. Theproximal and distal extent of vector delivery as determined byultrasound is marked with an indelible radiographic marker for referenceat the time of post-gene transfer muscle biopsy.

Subjects are followed with close monitoring of vital signs. Concomitantmedications are monitored and documented after injection. Subjects aredischarged two days after gene transfer (if no side effects areobserved).

Subjects return for follow up visits. Muscle biopsies are conducted atday 45 or day 90. Immune studies at 45 and 75 days post-gene transferand at 9, 12, 18, and 24 months include testing for binding antibody torAAVrh74 and antibody to GALGT2, as well as ELISpot to detect T cellresponse to capsid antigens. Subjects are seen at the end of first andsecond years for a physical exam, strength testing and immune studies.

Example 3 Efficacy Outcome Measurements

Muscle biopsies are taken from EDB muscles. Samples are coded tomaintain a blind in all subsequent analysis as to which was injectedwith rAAVrh74.MCK.GALGT2.

Efficacy outcome measures include: expression of GALGT2 as demonstratedwith anti-CT epitope antibodies; GALGT2 protein expression quantified bywestern blot and assessed by densitometry; transduction efficiencymeasured by qPCR of the GALGT transgene from muscle, and expressed asvector genomes normalized to a genomic single-copy control; the numberof fibers containing central nuclei compared between muscles by pairedt-tests; and analyses will also include: Dystrophin expression (withantibodies to N-terminal, C-terminal, and rod domains), utrophinexpression, and leukocyte markers including CD45, CD3, CD4, CD8, and MAC387. Muscle is examined for histological appearance. Antibodies torAAVrh74 along with PBMC ELISpots to both rAAVrh74 capsid and GALGTprotein are evaluated at different time points during the study up totwo years. The muscle analysis of gene expression and inflammation isalso done without breaking the blind.

Transgene expression is compared blindly between both EDB muscles from asingle subject, and between those subject's biopsies at day 45 or day90. A vector-specific primer probe set is used to amplify a unique 5′untranslated leader sequence of the transgene that will distinguishtransgene expressed GALGT2 protein from endogenous GALGT2.Quantification of protein is done using direct immunofluorescence (IF)and Western blot (WB) studies of muscle tissue. CD4+ and CD8+mononuclear cells are quantified by immunostaining and reported asnumber of cells/mm2 area. MHCI and MHCII antigen expression are assessedon muscle sections. Muscle morphometrics are also be performed,including fiber size histograms and quantification of centralnucleation. Analysis also includes PCR analysis for viral DNA.

Immune responses are assessed by IFN-γ ELISpots to GALGT2 and AAVcapsid. A rise in IFN-γ of >2SD to either virus or transgene isconsidered significant. An additional measure of immune response is thebinding antibody assay to AAV.

Measurements for improvements in one or more of absolute muscle specificforce; force decrement during eccentric muscle contractions; serum CKlevel; serum cardiac troponin level; serum MMP9 level; grip strength;limb torque; limb mobility or flexibility; ambulation; 6 minute walktest; knee flexor or extensor strength; maximal voluntary isometricmuscle contraction; North Star Ambulatory Assessment; muscle mass, fatreduction, or edema by limb T2-weighted MRI measures; musclecontractures; limb joint angle; heart function (heart rate, cardiacoutput, percent fractional shortening, stroke volume); respiration(including respiratory rate, blood oxygenation, need for supplementaloxygen); muscle necrosis; muscle regeneration; muscle wasting; muscleinflammation; muscle calcification; muscle central nucleation; musclesize or myofiber size; lifespan; and dystrophin or laminin alpha 2surrogate protein expression (utrophin, plectin 1, laminin alpha 5,agrin) are among those contemplated. See, for example, Forbes et al.,Radiology, 269(1): 198-207 (2013); Govoni et al., Cell Mol. Life Sci.,70(23): 4585-4602 (2013); and Chandrasekharan and Martin, MethodsEnzymol., 479: 291-322 (2010).

For each of the measures, statistical analysis based on differencesbetween pre- and post-gene transfer examinations (clinical, or on musclebiopsy) will be analyzed using a paired t test, with a p value of <0.05indicating significance.

Example 4 Vascular Delivery by Isolated Limb Perfusion

A vascular delivery route termed isolated limb perfusion (ILP) is alsocontemplated for treatment of human patients. Multiple leg muscles canbe targeted by ILP via delivery through the femoral artery. The methodpermits isolation of the limb from the general circulation, increasingtransduction efficiency and preventing virus from escaping to thegeneral circulation. [Rodino-Klapac et al., Mol. Ther., 18: 109-117(2010)]. An exemplary clinical protocol is set out below.

The rAAVrh74.MCK.GALGT2 is prepared as described in Examples 1 and 2.

The subject receives a stable dose of corticosteroid therapy (eitherprednisone or deflazacort, or their generic forms) for twelve weeksprior to treatment. Prednisone treatment is also continued after genetransfer. The sedated and anesthetized subject is secured to a surgicalbed. Proximal and distal tourniquets are loosely positioned above theknee and below the gastrocemius muscle of a macaque. A small incision isplaced at the femoral triangle and the femoral artery is identified anddissected free and looped with proximal and distal ligatures to controlbleeding and facilitate catheter introduction. The femoral artery iscannulated with a 3.0 Fr introducer sheath via a modified Seldingermethod by passing the pre-flushed sheath over a wire previously placedin the artery. The sheath is advanced only a few centimeters and securedin place with a 3.0 braided silk suture.

Following sheath placement in the femoral arteries and veins, 100-200u/kg of unfractionated heparin is administered and allowed to circulatefor 3-5 minutes. A Choice PT coronary guide wire is then placedinitially into the right femoral vein and artery, and then ultimatelyinto the left femoral vein and artery. A 4-mm diameter Tyshak MiniBalloon catheter is passed through the 3.3-French sheath through theright femoral artery into position in the femoral iliac artery junction.An 8-mm diameter×2 cm long Tyshak Mini Balloon catheter is passedthrough the 4-French sheath into appropriate position in the rightfemoral-iliac vein junction. Small hand injections of diluted contrastare performed to confirm appropriate blockage of both the left femoralartery and the right femoral vein. If needed, sheaths and balloons canbe exchanged for larger sizes. For example, the 4-French sheath in theright femoral vein can be exchanged for a 6-French sheath and a 12 mm×2cm long Tyshak II Balloon catheter can be passed over the Choice PTguidewire into appropriate position. Small hand injections through theside arm of the sheath are performed to confirm location and completeocclusion of the femoral vein.

A pre-flush of 2 mL/kg of Ringer's lactate heparinized solution isinfused after both right femoral artery and femoral venous balloons areinflated, with isolation of the right leg. After 1 minute, the Ringer'slactate flush at 2 mL/kg is completed. Next, the rAAVrh74.MCK.GALGT2vector is infused at a dose of between 2×10¹² vg/kg/limb and 4.8×10¹³vg/kg/limb in a volume of 8 mL/kg LR over 1½ minutes (since bilaterallimb perfusion is performed, leading to a total patient dose of between4×10¹² vg/kg and 9.6×10¹³ vg/kg). After the rAAVrh74.MCK.GALGT2 isdelivered, there is 10 minutes of dwell time, and then the right femoralarterial sheath is then used to infuse 2 mL/kg of heparinized Ringer'slactate over 1 minute. The balloons are then deflated, and the cathetersand guidewires are removed.

The left leg is then targeted for the same procedure. The left femoralartery is maintained with 3.3-French sheaths. Again, using a 4-mmdiameter Tyshak Mini Balloon catheter in the left femoral artery overthe Choice PT coronary guidewire, as well as the 12 mm×2 cm long TyshakII Balloon catheter through the 5-French sheath in the left femoral veinwith inflations up to 3 atmospheres of pressure, appropriate occlusionis demonstrated. The infusion protocol is repeated with 2 mL/kg ofheparinized Ringer's lactate infused over 1 minute, and a dose ofbetween 2×10¹² vg/kg/limb and 4.8×10¹³ vg/kg/limb of rAAVrh74.MCK.GALGT2is infused over 1 minute and 15 seconds, with the dwell time of 10minutes. Finally, 2 mL/kg of heparinized Ringer's lactate is infusedthrough left femoral arterial sheath, and then the sheaths are removedfrom all 4 access sites with pressure hemostasis and a HemCon patch.

Variations of this protocol can be used to deliver rAAVrh74.MCK.GALGT2via other arteries to alternative groups of muscles as needed. Forexample, delivery via the phrenic, intercostal and/or subcostal arteriesto supply the diaphragm muscle, or delivery via the coronary arteries tosupply the heart are contemplated. Similar doses would be utilized foreach procedure, and that multiple procedures might be done in a singlepatient or even in a single patient admission.

At the completion of dosing the tourniquets and catheter are removed anddirect pressure is applied to the wound for 10 min to control bleeding.The wound is closed with a continuous subcuticular 4.0 Vicryl suture. Apressure dressing is applied to the site and kept in place until thesubject awoke from anesthesia.

Following the ILP vector delivery protocol, subject follow up andefficacy outcome measurements/analyses similar to that described abovefor the IM-treated subjects are conducted.

Example 5 Systemic Vascular Delivery

Another contemplated route of delivery of the rAAVrh74.MCK.GALGT2 vectorto muscle is systemic vascular delivery. An exemplary dose escalationstudy examining efficacy can be conducted as follows.

Determination of Dose Range

IV injection (via the tail vein) of 1.4×10¹⁵ vg/kg rAAVrh74.MCK.GALGT2at day 1 of age causes transduction of over 90% of all limb skeletalmuscles in a wild type mouse, including tibialis anterior,gastrocnemius, quadriceps and triceps, and the same does leads to over50% transduction of all cardiomyocytes in the wild type mouse heart andover 70% of cardiomyocytes in the heart of mdx model mice heart.Notably, analysis of overall mdx mouse heart function at 3 months aftertreatment, relative to mock-treated mdx control animals, showed almost adoubling of cardiac output as the result of rAAVrh74.MCK.GALGT2treatment with this dose of vector, either with or without stimulationwith dobutamine, a beta agonist that stimulates heart rate. Thus, thereis an 80% increase in blood flow from the dystrophin-deficient heartafter rAAVrh74.MCK.GALGT2 treatment when vector is given prior to theonset of disease-related cardiac pathology. 5×10¹⁵ vg/kg is contemplatedto be the maximal therapeutic dose to transduce all heart and skeletalmuscle cells throughout the entire body using intravenous injection.Thus, it is contemplated that a dose range of about 5×10¹³ vg/kg toabout 5×10¹⁵ vg/kg would cover the minimally effective dose and theoptimally effective dose for rAAVrh74.MCK.GALGT2 treatment of the wholepatient in a clinical IV study.

Protocol

The rAAVrh74.MCK.GALGT2 is prepared as described in Examples 1 and 2.

The subject is started on prophylactic enteral prednisolone(glucocorticoid) (approximately 1 mg/kg/day) one day prior to therAAVrh74.MCK.GALGT2 administration. Prednisone treatment is alsocontinued after gene transfer.

On the day of gene transfer (Day 0) prior to rAAVrh74.MCK.GALGT2infusion, a physical exam is performed with vitals collected.

If a subject appears inadequately hydrated in the judgment of the PI,bolus(es) of 10-20 mL/kg normal saline may be given during the timebetween hospital admission and gene transfer. Subjects maintain theirusual diet until eight hours prior to gene transfer, after which theyhave no solid food; clear liquids are allowed up until two hours priorto gene transfer, after which they will be fully NPO. They resume theirusual PO intake after they return to pre-sedation baseline. Genetransfer will be performed under sterile conditions, under light tomoderate sedation under the direction of a qualified anesthesiologist.Sedation may vary, but the subject can be sedated using inhaled nitrousprior to induction with propofol via an IV, and maintained with inhaledsevoflurane or a propofol drip. In those subjects who, in the opinion ofthe PI (and in consultation with the anesthesiologist), are determinedto not need sedation in order to safely deliver the vector, sedation maybe deferred.

All subjects in the trial receive an intravenous injection ofrAAVrh74.MCK.GALGT2 via peripheral limb vein. The dose rangecontemplated is between 5×10¹³ vg/kg and 5×10¹⁵ vg/kg.

As one example, each vector dose is given undiluted, divided into 50 mLor less, to fill Becton Dickinson 60 mL capacity polypropylene syringes,prepared by the NCH Investigational Drug Pharmacy. The vector saltsolution is approximately 400 mOsmol/L. Infusion is performed using aSmiths Medical Medfusion 4000 Syringe Infusion Pump with PharmGuardInfusion Management Software Suite, delivered via a Smiths Medical MX563infusion tube. The infusion rate is not to exceed 2 ml/kg/min for anysubject. The infusion is given over approximately 10 to 20 minutes. Thevector is flushed from the infusion tubing using normal saline at theend of the infusion. It is contemplated that vector doses can be dividedand administered differently as necessary.

Subjects are closely monitored for side effects during the infusion,including continuous heart rate, respiratory rate, and pulse oximetry;and intermittent blood pressure monitoring. Heart rate, respiratoryrate, pulse oximetry, temperature, and blood pressure are measuredbefore and immediately after the infusion, and every five minutes duringthe infusion, and repeated at 15 minutes post-infusion.

Subjects remain in an intensive care unit bed following gene transferand remain admitted to the hospital for 48 hours after gene transfer.Vital signs are obtained hourly for 4 hours following the injection andthen every 4 hours until discharge. Transfer out of intensive care maybe undertaken after the initial 24 hours of post-infusion monitoring, ifthe PI has no concerns.

Following the vector delivery protocol, subject follow up and efficacyoutcome analyses similar to that described above for the IM-treatedsubjects are conducted.

While the present invention has been described in terms of specificembodiments, it is understood that variations and modifications willoccur to those skilled in the art. Accordingly, only such limitations asappear in the claims should be placed on the invention.

All documents referred to in this application are hereby incorporated byreference in their entirety with particular attention to the content forwhich they are referred. Also, this application claims the benefit ofthe filing date of U.S. Provisional Application Nos. 62/220,107 filedSep. 17, 2015; 62/221,068 filed Sep. 20, 2015 and 62/301,260 filedFebruary 2016; which are incorporated by reference in their entiretyherein.

1-8. (canceled)
 9. A method of treating a neuromuscular disorder in ahuman subject in need thereof comprising the step of administering tothe human subject a recombinant adeno-associated virus (rAAV), whereinthe rAAV comprises a genome comprising a nucleic acid comprising, inorder from 5′ to 3′: (i) a first AAV2 inverted terminal repeat sequence(ITR); (ii) an MCK enhancer comprising nucleotides 236-442 of SEQ ID NO:2; (iii) a muscle creatine kinase core promoter sequence comprisingnucleotides 443-793 of SEQ ID NO: 2; (iv) a nucleotide sequence encodinga human β1-4-N-acetyl-D-galactosamine glycosyltransferase (GALGT2)polypeptide; and (v) a second AAV2 ITR sequence; wherein the humanGALGT2 polypeptide has an amino acid sequence that is at least 90%identical to SEQ ID NO: 3 or is 100% identical to SEQ ID NO: 3, or isencoded by a nucleotide sequence 90% identical to nucleotides 1002-2522of SEQ ID NO: 2 or 100% identical to nucleotides 1002-2522 of SEQ ID NO:2.
 10. The method of claim 9 wherein the route of administration is anintramuscular route and the dose of the rAAV administered is about3×10¹¹ vector genomes/injection to about 5×10¹² vg/injection, anintramuscular route and the dose of the rAAV administered is about3×10¹¹ vector genomes/injection, an intramuscular route and the dose ofthe rAAV administered is about 1×10¹² v vector genomes/injection, anintramuscular route and the dose of the rAAV administered is about5×10¹² vector genomes/injection, inter-arterial limb perfusion and thedose of the rAAV administered is about 6×10¹² vector genomes/kg/limb toabout 4.8×10¹³ vector genomes/kg/limb, inter-arterial limb perfusion andthe dose of the rAAV administered is about 6×10¹² vectorgenomes/kg/limb, inter-arterial limb perfusion and the dose of the rAAVadministered is about 1.2×10¹³ vector genomes/kg/limb, inter-arteriallimb perfusion and the dose of the rAAV administered is about 2.4×10¹³vector genomes/kg/limb, inter-arterial limb perfusion and the dose ofthe rAAV administered is about 4.8×10¹³ vector genomes/kg/limb, systemicintravenous administration and the dose of the rAAV administered isabout 2×10¹⁴ vector genomes/kg to about 6×10¹⁵ vg/kg, systemicintravenous administration and the dose of the rAAV administered isabout 4×10¹⁴ vector genomes/kg to about 6×10¹⁵ vector genomes vectorgenomes/kg, systemic intravenous administration and the dose of the rAAVadministered is about 4×10¹⁴ vector genomes/kg, systemic intravenousadministration and the dose of the rAAV administered is about 8×10¹⁴vector genomes/kg, systemic intravenous administration and the dose ofthe rAAV administered is about 2×10¹⁵ vector genomes/kg, or systemicintravenous administration and the dose of the rAAV administered isabout 6×10¹⁵ vector genomes/kg.
 11. The method of claim 9, wherein theneuromuscular disorder is Duchenne Muscular Dystrophy (DMD); BeckerMuscular Dystrophy; Congenital Muscular Dystrophy (MDC) 1A, 1B, 1C and1D; Limb Girdle Muscular Dystrophy (LGMD) 1A, 1B, 1C, 1D, 1E, 1F, 1G,1H, 2A, 2B, 2C, 2D, 2E, 2F, 2G 2H, 2I, 2J, 2K, 2L, 2M, 2N, 20 and 2Q;Bethlem Myopathy; Ullrich Congenital Muscular Dystrophy; Muscle EyeBrain Disease; Fukuyama Congenital Muscular Dystrophy; Walker WarburgSyndrome; Myotonic Dystrophy; Myasthenic syndromes; CongenitalMyasthenias; Inclusion Body Myopathy; Inclusion Body Myositis; EmeryDreifuss Muscular Dystrophy; Distal Muscular Dystrophy; Dermatomyositis;Centronuclear Myopathy; Faciosacpulohumeral Muscular Dystrophy; MyoshiMyopathy; Mitochondrial Myopathy; Nemaline Myopathy; Nonaka Myopathy;Myasthenia Gravis; and Polymyositis.
 12. The method of claim 9 wherebythere is an improvement in the human subject in absolute muscle specificforce; force decrement during eccentric muscle contractions; serum CKlevel; serum cardiac troponin level; serum MMP9 level; grip strength;limb torque; limb mobility or flexibility; ambulation; 6 minute walktest; knee flexor or extensor strength; maximal voluntary isometricmuscle contraction; North Star Ambulatory Assessment; muscle mass, fatreduction, or edema by limb T2-weighted MRI measures; musclecontractures; limb joint angle; heart function (heart rate, cardiacoutput, percent fractional shortening, stroke volume); respiration(including respiratory rate, blood oxygenation, need for supplementaloxygen); muscle necrosis; muscle regeneration; muscle wasting; muscleinflammation; muscle calcification; muscle central nucleation; musclesize or myofiber size; lifespan; and dystrophin or laminin alpha 2surrogate protein expression (utrophin, plectin 1, laminin alpha 5,agrin).
 13. The method of claim 9, wherein the nucleic acid furthercomprises 3′ to said core promoter, a mouse MCK exon 1 sequencecomprising nucleotides 794-846 of SEQ ID NO:
 2. 14. The method of claim13, wherein the nucleic acid further comprises 3′ to said core promoter,an SV40 intron sequence comprising set out in nucleotides 847-943 of SEQID NO:
 2. 15. The method of claim 14, wherein the nucleic acid furthercomprise 3′ to said core promoter, a 5′ untranslated region comprisingset out in nucleotides 944-1000 of SEQ ID NO:
 2. 16. The method of claim15, wherein the nucleic acid further comprises 3′ to said nucleotidesequence encoding a human GALGT2 polypeptide, a Syn pA syntheticpolyadenylation signal sequence comprising set out in nucleotides2531-2579 of SEQ ID NO:
 2. 17. The method of claim 9, wherein in thenucleic acid said first ITR comprises nucleotides 53-230 of SEQ ID NO:2, and/or said second ITR comprises nucleotides 2581-2762 of SEQ ID NO:2.
 18. The method of claim 16, wherein in the nucleic acid said firstITR comprises nucleotides 53-230 of SEQ ID NO: 2; and said second ITRcomprises nucleotides 2581-2762 of SEQ ID NO:
 2. 19. The method of claim9, wherein the rAAV comprises an rAAV.rh74.MCK.GALGT2 genome that is atleast 90% identical to the nucleotide sequence of set out in SEQ ID NO:2.
 20. A method of claim 9, wherein the AAV comprises therAAV.rh74.MCK.GALGT2 genome set out in SEQ ID NO:
 2. 21. The method ofclaim 9, wherein the rAAV is administered as is a recombinantadeno-associated virus particle.
 22. The method of claim 9, wherein therAAV is serotype AAV-1, AAV-2, AAV-3, AAV-4, AAV-5, AAV-6, AAV-7, AAV-8,AAV-9, AAV-10, AAV-11, or AAVrh.74.
 23. The method of claim 9, whereinthe AAV DNA in the rAAV genome is from AAV rh.74.
 24. The method ofclaim 24, wherein the polynucleotide sequence of the AAV rh.74 genomecomprises the nucleotide sequence of SEQ ID NO: 1.